Chronic Hepatitis C virus (HCV) infection is a leading cause of cirrhosis, end-stage liver disease and liver cancer in the USA. There is no vaccine and until 2011, the standard of care was dual antiviral therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV). PEG-IFN/RBV therapy is poorly tolerated and only leads to sustained virological response (SVR) in 50% of patients infected with genotype (gt) 1a HCV, the most commonly found gt in the USA. To address this problem, intensive efforts were made to identify small molecule inhibitors targeting HCV. As a result of this research, many direct-acting antivirals (DAA) have been identified and several are in clinical development. The past three years have seen the approval of 5 new DAAs for the treatment of chronic hepatitis C including small molecules specifically designed to target the HCV protease and polymerase enzymes. These drugs improve SVR rates but must be administered in combination with other DAAs or with PEG-IFN/RBV to avoid problems of drug resistance emergence. Inhibitors of the HCV protease and polymerase enzymes that are currently in clinical development were designed based on a detailed knowledge of structure-activity relationships. Another class of antivirals in clinical development is the NS5A inhibitors. Unlike protease and polymerase inhibitors, NS5A inhibitors were identified by cell-based screening assays for compounds with anti-HCV activity. NS5A was subsequently identified as the target for this class of compounds based on sequence analysis of resistant variants. NS5A inhibitors in clinical development will form a key component of future interferon-free DAA combinations for chronic hepatitis C therapy. Indeed the first interferon-free, all oral therapy for chronic hepatitis C was approved in October 2014 and comprises a polymerase inhibitor in combination with an NS5A inhibitor. The HCV NS5A protein is unique to HCV and related viruses, it has no enzymatic activity and limited structural information is available. Experimental data suggest it acts in multiple aspects of the HCV lifecycle. However NS5A remains poorly characterized and how its normal functions are affected by NS5A inhibitors is not well understood. In the proposed studies, I will study the mechanism of action of NS5A inhibitors using H77S.3, a cell culture infectious gt 1a strain of HCV. Specifically, I will determine how NS5A inhibitors affect virus RNA synthesis, virus assembly and egress. At the molecular level, quantitative proteomic approaches will be employed to determine how NS5A inhibitors affect NS5A interactions with cellular and viral proteins to block virus assembly. I will examine how inhibitors affect the subcellular localization of NS5A with respect to interaction partners and also intracellular membrane structures at high spatial resolution. These studies will reveal mechanistic details underlying the action of this potent new class of antiviral.